Fig. 2. Bacteria allocate resources to glycerophospholipid metabolism to support CST resistance.

(A) Percent survival of Ec-MCR-1 cells in the presence of EA (0, 0.5, 1, and 2 mM) and CST (2 μg/ml) (n = 4) for 6 hours. Con, Control. (B) Percent survival of Ec-MCR-1 cells in the presence of EA (0 and 2 mM) and indicated concentrations of CST for 6 hours (n = 4). (C) Colony-forming units (CFUs) of Ec-MCR-1 cells in the presence of EA (1 mM) or l-arabinose (Ara) (10 mM) or both at indicated incubation times (n = 3). (D) Specific enzyme activities of PDH, OGDH, SDH, and MDH in Ec-MCR-1 cells in the presence of EA or PE (n = 4). (E) Schematic of glycolysis and glycerophospholipid metabolism connection. (F) mRNA levels of glycerophospholipid metabolism genes in Ec-K12, Ec-MCR-1, and Ec-E246A (n = 5). (G) Percent survival of Ec-MCR-1 and Ec-MCR-1::ΔpssA in the presence of PE (2 mM) and/or CST (4 μg/ml) for 6 hours (n = 4). (H) PDH, OGDH, SDH, and MDH activities in the indicated bacterial strains (n = 4). (I) Percent survival of Ec-MCR-1 cells in the presence of the indicated concentrations of Barlox 12 and CST for 6 hours (n = 4). (J) Glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) abundance in Ec-MCR-1 and Ec-E246A cells (n = 4). (K) Specific enzyme activities of 6-phosphofructokinase (PFK) and pyruvate kinase (PK) in Ec-MCR-1 and Ec-E246A (n = 4). (L) Percent survival of indicated mutant bacteria in the presence of CST (2 μg/ml) (n = 4). Specific enzyme activity was measured in whole-cell lysates and normalized to total protein abundance [(D), (H), and (K)]. Percent survival was calculated by dividing the CFU of the treated group by that of the nontreated group. Data are presented as mean ± SD [(A) to (D) and (F) to (L)]. Statistical significance was assessed using Student’s t test, with a P value threshold of 0.05 (P < 0.05) considered statistically significant. n denotes biological replicates.